The long-term objectives of the proposed research are to expand present knowledge of the cellular mechanisms whereby hormones and ions regulate mammalian renal proximal tubule synthesis of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). 1,25(OH)2D3 is the major regulator of intestinal Ca and P absorption. 1,25(OH)2D3 synthesis varies during the life-cycle, increasing during growth and decreasing in the elderly, to meet bone Ca and P requirements. Integration of bone mineral requirements and renal 1,25(OH)2D3 synthesis remains unknown, but parathyroid hormone (PTH), insulin-like growth factor I (IGF-1) and low P diet (LPD) are major recognized stimuli of the renal mitochondrial 25-hydroxyvitamin D3-1 alpha hydroxylase (1-OHase). In proximal tubules, PTH activates both the cAMP/cAMP-dependent protein kinase (PKA) and the phospholipase C (PLC)/protein kinase C (PKC) signalling systems. Although either system could mediate PTH action on 1-OHase, PTH activates PKC and stimulates 1,25(OH)2D3 at the same physiologic concentration, whereas greater concentrations of PTH are required for cAMP/PKA activation. The mechanism whereby IGF-1 and LPD stimulate 1-OHase remains unknown, but IGF-1 may facilitate 1-OHase response to PTH and LPD. The current application proposes to test the overall hypotheses that the PLC/PKC system mediates PTH activation of 1-OHase and that systemic and/or renal paracrine IGF-1 amplifies 1-OHase activity during PTH and LPD. The hypotheses will be tested by the following Specific Aims: 1) to identify the PKC isoforms involved in acute and chronic PTH stimulation of rat proximal tubule 1,25(OH)2D3 production; 2) to determine whether net 1,25(OH)2D3 production by PTH, IGF-1 and LPD results from reciprocal stimulation of 1-OHase and suppression of 24- OHase activities; 3) to explore potential mechanisms whereby IGF-1 stimulates proximal tubule 1-OHase activity; and 4) using knowledge from the above studies, to investigate the mechanism for reduced 1,25(OH)2D3 production during aging. The proposed studies are intended to improve the understanding of physiologic control of 1,25(OH)2D3 and defective 1,25(OH)D3 production during aging and diseases of bone loss and fracture, such as osteoporosis and primary hyperparathyroidism.